The DNA mismatch repair (MMR) system corrects DNA synthesis errors that occur during replication and is also involved in several other DNA transactions. MMR is initiated by MutS and MutL homologs, which are highly conserved throughout prokaryotes and eukaryotes. They are both dimers and contain DNA binding and ATPase activities that are essential for MMR in vivo. Inactivation of these proteins leads to increased mutagenesis, improper recombination, and resistance to the cytotoxic effects of several DNA damaging agents. In humans, mutations in the mismatch repair genes are directly linked to hereditary non-polyposis colorectal cancer (HNPCC) and are associated with several sporadic cancers. Because of the diversity of functions carried out by the MMR proteins, it will be essential to understand the molecular mechanisms that underlie these different processes to develop effective treatment for the associated diseases and cancers. In eukaryotes, MutS? (MSH2-MSH6) and MutL? (MLH1-PMS2, Mlh1-Pms1 in yeast) are the primary MutS and MutL homologs responsible for initiation of MMR. MutS? initiates repair by binding to a mismatch and undergoing an ATP-dependent conformational change that promotes its interaction with MutL?. PCNA then activates MutL? to incise the daughter strand both 5' and 3' to the mismatch. Subsequently, MutS? activates the 5'-3' exonuclease EXO1 to processively excise the DNA containing the incorrect nucleotide. Finally, DNA polymerase ? or ? catalyzes resynthesis, and DNA ligase seals the nick. Structural and biochemical studies, including several from our labs, indicate that the conformational dynamics and assembly states of the proteins and protein-DNA complexes are central to the regulation of MMR. The overall goal of this proposal is to elucidate the structure/function relationships that govern the initiation of MMR in vivo. We propose a systematic series of experiments that bring our sensitive single-molecule fluorescence methods to live cells using S. cerevisiae as a model system. We will take advantage of both established technologies, such as fluorescent proteins, as well as rapidly emerging technologies, such as unnatural amino acid labeling. To bring this project to fruition, we have assembled a team with both strong expertise in every aspect of the project and a strong penchant for collaborative work. In addition, our collaboration with Dr. Thomas Kunkel significantly strengthens our ability to carry out the project as well as to test the biological significance of the models that result. ur goals are 1) to determine the molecular compositions of in vivo mismatch repair complexes, including stoichiometries and relative locations of proteins, using single-molecule fluorescence coupled with super-resolution techniques, and 2) examine the in vivo conformational dynamics of the mismatch repair initiation proteins, MutS? and MutL?, during repair using single-molecule FRET. The proposed experiments will answer many outstanding questions about the molecular mechanisms of MMR, and they will expand the range of techniques for examining the in vivo dynamics and compositions of multiprotein complexes.